All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Hematopoietic stem cells (HSCs) are capable of self-renewal and are able to give rise to all blood cell lineages. These cells, which reside in the bone marrow, form the basis of the adult hematopoietic system. The transplantation of these cells represents the most common cell-based therapy applied today in the clinic.
HSC transplantation is used for the treatment of patients with acute or chronic leukemia, aplastic anemia and various immunodeficiency syndromes, as well as various non-hematological malignancies and auto-immune disorders. For patients with hematological malignancies, HSC transplantation may rescue patients from treatment-induced aplasia, which can occur following high-dose chemotherapy and/or radiotherapy.
Despite the widespread clinical utility of HSC transplantation, the three major sources of HSCs (human bone marrow, mobilized peripheral blood, and umbilical cord blood) are limited as two-thirds of the patients in need of somatic HSC transplantation lack well-matched donors. For example, for any given patient, there is only a 25% chance that a sibling is a human leukocyte antigen (HLA)-identical match.
HLAs are proteins on a cell's surface that help the immune system identify the cells as either self (belonging to the body) or non-self (foreign or from outside the body). The HLA proteins are encoded by clusters of genes that form a region located on human chromosome 6 known as the Major Histocompatibility Complex, or MHC, in recognition of the important role of the proteins encoded by the MHC loci in graft rejection. Accordingly, the HLA proteins are also referred to as MHC proteins. Although matching the MHC molecules of a transplant to those of the recipient significantly improves the success rate of clinical transplantation, it does not prevent rejection, even when the transplant is between HLA-identical siblings. Such rejection may be triggered by differences between the minor Histocompatibility antigens. These polymorphic antigens are usually “non-self” peptides bound to MHC molecules on the cells of the transplant tissue, and differences between minor Histocompatibility antigens will often cause the immune system of a transplant recipient to eventually reject a transplant, even where there is a match between the MHC antigens, unless immunosuppressive drugs are used.
There are three types each of class I and class II HLA. A person (typically a sibling) who has a class I and class II HLA match is called a related donor. Increased survival is associated with a match between recipient and donor class I HLA-A, HLA-B, HLA-C, and class II HLA-DRB1 and HLA-DQB1 (Morishima, et al., 2002 Blood (99):4200-6). For a patient who does not have a matched, related donor, a search through donor banks may provide a person with matching HLA types. However the number of people in need of a cell or tissue transplant, such as an HSC transplant, is far greater than the available supply of cells and tissues suitable for transplantation. Under these circumstances, it is not surprising that obtaining a good match between the MHC proteins of a recipient and those of the transplant is frequently impossible. Therefore, many transplant recipients must wait for an MHC-matched transplant to become available or accept a transplant that is not MHC-matched and endure higher doses of immunosuppressive drugs and still risk rejection. The ability to generate and manipulate HSCs, and/or to induce tolerance in recipients of transplants, therefore, will greatly benefit the treatment and management of human disease.
Based on work in the avian embryo, and subsequently in frogs and mammals, it has been demonstrated that the developmental programs of blood and endothelium are closely linked. For example, endothelial and hematopoietic cells emerge concurrently and in close proximity in yolk sac blood islands. The yolk sac blood islands derive from aggregates of mesodermal cells that colonize the yolk sac. The center of these aggregates gives rise to the embryonic hematopoietic cells whereas the peripheral population differentiates into endothelial cells which form the vasculature that surrounds the inner blood cells. These observations support the notion that endothelial and hematopoietic cells have a common precursor.
Additionally, in zebrafish and mouse embryos, both endothelial and hematopoietic lineages share the expression of certain genes, such as Flk1, Flt1, Tie1, Tie2, CD34, Scl, and Runx1 (Fina et al. 1990 Blood (75): 2417-2426; Millauer et al. 1993 Cell (72): 835-846; Yamaguchi et al. 1993 Development (118): 489-498; Anagnostou et al. 1994 PNAS USA (91): 3974-3978; Kallianpur et al. 1994 Blood (83): 1200-12081; Young et al. 1995 Blood (85): 96-105, Asahara et al. 1997 Science (275): 964-967; Kabrun et al., 1997 Development (124): 2039-2048). Likewise certain gene mutations affect both endothelial and hematopoietic cell development (Shalaby et al. 1995 Nature (376): 62-66; Robb et al. 1995 PNAS USA (92): 7075-7079; Shivdasani et al. 1995 Nature (373): 432-434; Stainier et al. 1995 Development (121): 3141-3150; Bollerot et al., 2005 APMIS (113): 790-803). Further, the deletion of either Flk1 or Flt1, which are both receptors for vascular endothelial growth factor (VEGF), results in a disruption of hematopoietic and endothelial development in the mouse embryo.
The generation of mouse hemangioblasts from mouse embryonic stem cells in vitro has been reported in the literature (Choi et al. 1998 Development 125: 727-732). Further, human precursor cells capable of giving rise to both hematopoietic and endothelial cells have been derived from human ES cells (Wang et al. 2004 Immunity (21): 31-41 and Wang et al. J Exp Med (201): 1603-1614), but only in small quantities, hundreds of cells at best. Moreover, no method or conditions exist for the expansion of the hemangioblast precursor cells in vitro.
Despite the prior identification of cell types capable of giving rise to cells of the hematopoietic lineage, there remains a need for improved methods of generating large numbers of progenitor cell populations, as well as differentiated cell types. The present invention addresses this need. Additionally, the present invention provides a novel progenitor cell type that does not engraft into the bone marrow when delivered in vivo to an immunodeficient animal. However, despite the inability of these cells to engraft into the bone marrow, the cells are capable of generating differentiated hematopoietic cells, and may even be capable of generating other non-hematopoietic cells types such as endothelial cell types, skeletal muscle, and smooth muscle.
There is additionally a critical need for available blood for transfusion. The Red Cross and other suppliers of blood report a near constant shortage of blood. This is especially true for patients with unique blood types, patients who are Rh+, or following accidents or disasters resulting in mass casualties. Additionally, in times of war, the military has an acute need for available blood for use in the treatment of traumatic war-related injuries. The present invention provides differentiated hematopoietic cells for use in blood banking and transfusion. The cells and methods of the present invention will provide a safe and reliable advance beyond the traditional reliance on blood donations, and will help prevent critical shortages in available blood.
Thus, there remains a need for methods for generating and expanding large numbers of human hemangioblasts as well as hemangioblast derivative cell types, i.e., hematopoietic and endothelial cells, and all solutions/mixtures containing such quantities of hemangioblasts or derivative cell types. Such methods would increase the availability of cells for transplantation as well as have utility in a variety of other therapeutic applications, such as in induction of immunological tolerance.